1. Field of the Invention
The present invention relates to a new class of fluorescent, calcium-binding probe compounds having visible-light excitation and emission wavelengths, and to the use of these probe compounds for making wavelength-ratiometric or intensity-ratiometric measurements of calcium ion concentrations in samples. The invention is particulary well-suited for making wavelength-ratiometric or intensity-ratiometric measurements of intracellular Ca.sup.2+ levels in which the Ca.sup.2+ measurements can be accomplished independent of the overall concentration of the probe compound.
2. Description of the Related Art
Calcium is a recognized intracellular messenger and mediator. Consequently, it is not surprising that many and varied proposals for measuring calcium ion concentrations have been proposed in the field for the purpose of facilitating the study of cell activity, such as cell response to a variety of stimuli.
To achieve this purpose, most proposals for measurements of intracellular Ca.sup.2+ use fluorescence indicators, such as Quin-2, Indo-1 and Fura-2, as described for example in U.S. Pat. No. 4,603,209 and Soc. Gen. Physiol. Ser., V. 40, 1986, pages 327-45.
However, it is difficult to design novel fluorophores which are both specific for calcium and which display fluorescence spectral shifts upon calcium binding. In fact, only a small fraction of the known chromophores display satisfactory fluorescent properties and chemical specificity in an aqueous solution.
Therefore, in spite of the past intense efforts to acquire suitable calcium ion probes, these efforts have been disappointing because of the severe limitations encountered with the heretofore proposed dyes.
For instance, the known dyes, such as Quin-2, require excitation in the UV wavelength range where autofluorescence from cells is excited. Further, the transmission of UV light in microscopic optics applications is deficient, as explained in U.S. Pat. No. 4,603,209. For example, microscope optics are traditionally corrected for chromatic aberration in the visible region of the spectrum. This creates problems in the area of confocal microscopy, which requires precise alignment of pinhole apertures for a range of excitation wavelengths.
In the case of existing wavelength-ratiometric calcium probes, these wavelengths are in the UV region, and the confocal measurements also fail because of uncorrected chromatic aberration in this wavelength region. Additionally, there are simpler and less expensive laser sources available for visible wavelengths rather than for UV wavelengths, which enable not only disc scanning confocal microscopy, but also laser scanning confocal microscopy.
Moreover, other known dyes, such as Fura-2 and Quin-2, are subject to photobleaching, and, perhaps, even more important, photoconversion to Ca.sup.2+ -insensitive forms. These insensitive dye forms are fluorescent and interfere with the determination of the Ca.sup.2+ concentration, as described in U.S. Pat. No. 5,049,673.
Therefore, because of these troublesome problems associated with the UV-excited dyes, there has been recognized a need for Ca.sup.2+ probes having improved photostability and longer wavelength absorption and emission. That is, longer wavelength absorption is desirable because the amount of autofluorescence decreases progressively as a function of increasingly longer wavelengths for excitation of the probe.
There exist in the prior art several proposals for satisfying this need. For instance, U.S. Pat. No. 4,849,362 describes longer wavelength tetracarboxylate compound chelators for calcium ions. The tetracarboxylate compounds in U.S. Pat. No. 4,849,362 are reported to experience an increase in fluorescence intensity upon excitation, and the optical responses reportedly could be determined by using a single visible wavelength laser line for excitation. However, the probes in U.S. Pat. No. 4,849,362 do not display any spectral shifts upon calcium binding and thus wavelength-ratiometric measurements are not possible with the dyes described in this patent.
That is, the field has not only appreciated the need to provide a longer-wavelength (visible light) fluorescent dye for making calcium ion measurements, but also has further envisaged the possible salutary benefits that could be obtained by the discovery of a fluorescent dye capable of enabling wavelength-ratiometric measurements of Ca.sup.2+ concentrations.
As understood in the field, the phrase "wavelength-ratiometric measurement" generally means a ratio of intensities measured at two different excitation or emission wavelengths. That is, a "wavelength-ratiometric measurement" means making a calcium concentration measurement in a sample by a process wherein the calcium ion-containing sample is excited at two different wavelengths, sequentially, and the resulting fluorescence intensities are measured for each excitation, and the ratio of the respective fluorescence intensities provides an indication of the calcium ion concentration in the sample when compared to a calibration curve.
This ratiometric measuring ability in a dye is highly desirable and often necessary because the probe concentration cannot be easily controlled (and/or the emitted fluorescence cannot be controlled) in flow cytometry, microscopy, or in cuvette studies involving cell suspensions.
The acute difficulties in developing a long-wavelength fluorescent dye which is concomitantly capable of providing such ratiometric measurements of calcium ions in intracellular samples is highlighted in recent U.S. Pat. No. 5,049,673 which proposes certain visible-wavelength fluorescent dyes of a RHOD series and a FLUO series.
However, the elusiveness of developing such long-wavelength probes that also permit ratiometric measurements is recognized in the U.S. Pat. No. 5,049,673. In this regard, U.S. Pat. No. 5,049,673 reports the inability to provide long-wavelength fluorophores which have wavelength pairs in either excitation or emission that are suitable and needed for fluorescence ratioing.
As stated in U.S. Pat. No. 5,049,673, ratioing would be extremely valuable with single cells because it cancels out variations in dye concentration and path length. Without such ratioing, it is impossible to correlate dye fluorescence intensity with Ca.sup.2+ levels in a simplified manner.
U.S. Pat. No. 5,049,673 concedes that the proposed dyes disclosed therein had only small or negligible shift in absorbance, excitation or emission wavelengths upon Ca.sup.2+ binding; consequently, such dyes of that patent are unsuitable for accomplishing fluorescence ratioing.
However, from a more general perspective, certain derivatives of BAPTA have been proposed as absorption and/or colorimetric indicators which are reported to be detectable at longer wavelengths which would shift to other wavelengths when complexed with calcium. This shifting would allow quantitative analysis for calcium without interference from UV and short-wavelength visible light-absorbing species, such as disclosed in U.S. Pat. No. 4,795,712.
However, U.S. Pat. No. 4,795,712 does not describe the ability of the dyes described therein to fluoresce, much less the application of such dyes to fluorescence detection. Instead, U.S. Pat. No. 4,795,712 is directed only to the use of certain chelating compounds to detect Ca.sup.2+ in liquids or spread (dry) samples based on changes in the visible absorbance and/or reflective spectra of samples containing these compounds.
In any event, U.S. Pat. No. 4,795,712 does not address the development of fluorescent dye compounds capable of permitting ratiometric measurements of calcium ions in intracellular environments using fluorescence detection.
Further, in general, it is known that the BAPTA chelating group is valuable in cellular systems because of its specificity for Ca.sup.2+ and low affinity for Mg.sup.2+, Na.sup.+ or K.sup.+ (R. Y. Tsien, Methods in Cell Biology, 1989, Academic Press, London; R. Y. Tsien, Biochem., 1980, 19: 2396-2404).
Another desirable feature of potential probes is the use of charge steering by cations, which are described heretofore using crown ether and azacrown moieties (H. G. Lohr and F. Vogtle, 1985, Acc. Chem. Res., 1985, 18: 65-72). According to the above-described prior art, there have been synthesized various probes which contain such substances as styryl dyes, various chromophores, and crown ethers to complex cations. These types of substances can display changes in their absorption spectra in response to cation binding. For example, see J. F. Alder, D. C. Ashworth, R. Narayanaswamy, R. E. Moss and I. O. Southerland, 1987, Analyst, 112: 1191-1192; S. Fery-Forgues, M. T. LeBris, J. P. Guette and B. Valeur, J. Chem. Soc., Chem. Commun. 1988, 385; J. Bourson and B. Valeur, J. Phys, Chem., 1989, 93: 3871-3876; S. Fery-Forgues, M. T. LeBris, J. P. Guette and B. Valeur, J. Phys. Chem., 1988, 92: 6233-6237; M. V. Alfimov, S. P. Gromov and I. K. Lednev, 1991, Chem. Phys. Lett., 185: 455-460.
However, these prior usages of crown ether chemistry resulted in low specificity and/or affinity for Ca.sup.2+, which effectively precludes the use of these probes in cellular systems. Additionally, these prior crown ether probes did not contain carboxylic acid groups or provide the opportunity for cell labeling via the acetoxymethylester-esterase trapping procedure. Also, the use of simpler fluorophores did not result in fluorescence spectral shifts (K. W. Street, Jr., Anal. Lett., 1986, 19: 735-745).
In view of the above-discussed technological backdrop, it is apparent that the field has urgently awaited the innovative development of long-wavelength fluorescent compounds which are concomitantly able to permit wavelength-ratiometric or intensity-ratiometric measurements of Ca.sup.2+ concentrations, especially intracellular calcium concentrations.